CN1239103A - Production method of recombinant human serum albumin - Google Patents

Abstract

The present invention relates to a method for producing human serum albumin by using recombinant DNA technology, in particular, it relates to a synthetic human serum albumin gene formed from yeast biased codon and undergone the process of proper modification, recombinant expression vector carrying said gene and yeast host cell transformed by said vector, and a method for producing human serum albumin by using said yeast host, in particular methyl nutrient yeast host in secretory mode.

Description

Production method of recombinant human serum albumin

The present invention relates to the method for producing human serum albumin by recombinant DNA technology, particularly relate to the synthetic human serum albumin gene that is composed of yeast partial codons and through appropriate modification, the recombinant expression vector that carries said gene and use A yeast host cell transformed with said vector, and a method for producing human serum albumin in a secretable form by said yeast host, especially a methylotrophic yeast host.

Human serum albumin (HSA) is the main constituent protein of human plasma, and its content accounts for more than 50% of the total plasma protein. Under normal human physiological conditions, human plasma albumin has the functions of maintaining plasma colloid osmotic pressure, nourishing and promoting wound healing, and as a carrier substance, it participates in many hydrophobic biomolecules such as hormones, biologically active substances and drugs. transport in the blood. Based on these important physiological functions of human serum albumin, the protein has very important therapeutic application value in clinic, and can be used to treat various diseases whose basic characteristics are the loss of plasma albumin or the reduction of albumin synthesis, such as the treatment of various trauma, burns, blood loss during or after surgery, and many chronic wasting diseases.

So far, clinically used HSA and related preparations are mainly obtained from blood donors or placental tissue through extraction and separation. The main disadvantage of using naturally extracted HSA is that, on the one hand, it is limited by the limited supply of blood, that is, limited blood sources are difficult to meet the needs of mass production of HSA and its related products; on the other hand, due to improper selection of blood donors, it may often cause Blood contamination, for example, hepatitis virus, or even AIDS virus contamination. Therefore, people have been trying to establish alternative methods for the commercial production of HSA. With the development of recombinant DNA technology, it is now possible to use this technology to produce HSA in large quantities.

Lawn et al. (Nucleic Acids Research9: 6105, (1981)) and Dugaiczyk et al. (Proc. Natl. Acad. Sci. USA79: 71, (1982)) isolated the gene encoding human serum albumin and determined its nuclear nucleotide sequence. According to the HSA amino acid sequence deduced from the measured nucleotide sequence, the mature HSA is composed of 585 amino acids, a single-chain non-glycosylated protein with a molecular weight of about 66,000 Daltons and an isoelectric point of Ip4.7. When the protein is first produced, it exists in the form of preproprotein, and the signal peptide and preprosequence are respectively removed during secretion and processing by cells to obtain a mature HSA molecule. The mature HSA molecule has an elliptical three-dimensional structure and contains multiple disulfide bonds. After the DNA coding sequence of HSA was isolated, many researchers attempted to mass-produce HSA with relatively high purity by recombinant DNA technology in various expression systems. For example, Barns described the isolation of HSA mRNA from normal human hepatocytes and the synthesis of full-length cDNA encoding pre-HSA using this mRNA as a template with the aid of reverse transcriptase, Klenow fragment and SI nuclease (see European Patent Application 0206733) , and described the plasmid carrying the cCDA and its expression in E. coli. However, since the molecular weight of human serum albumin is relatively large and the configuration is complex, prokaryote Escherichia coli (Latta, M.etal., Bio/Technology, 5:1309-1314, (1987)) and Bacillus subtilis ( Prokaryotic expression systems such as Saunders, C.W.et al, J.Bacteriol.169:2917-2925, (1987)) are difficult to obtain correctly folded expression products. In addition, it is also difficult to obtain HSA products suitable for clinical application in large doses due to the presence of bacterial endotoxins and other protein contaminants that are difficult to remove in the secreted expression products.

Mammalian cells such as CHO cells, COS cells, and insect cell expression systems can produce protein products with correct spatial configurations, but the nutritional conditions for cultivating these cells are relatively high, and their passage time is much longer than that of microorganisms, so that they must undergo It takes a long time to culture to reach a sufficiently high cell concentration, and it is generally difficult to make the necessary modifications to these cells. Therefore, the use of these eukaryotic organisms for the production of HSA is not economical.

Since all eukaryotic organisms have mechanisms for expressing genetic information, it is expected that eukaryotic genes can be expressed more efficiently in eukaryotic hosts than in prokaryotic organisms such as E. coli. In recent years, some laboratories have made breakthroughs using lower eukaryotic organisms, especially yeast, to express human serum albumin. For example, European patent application 0344459 describes the construction of a plasmid containing the transcription unit, which carries the HSA gene under the control of the AOXI promoter and is introduced into a suitable host, in particular the AOX1 gene region of a Pichia strain (GTS115) to obtain a transformant cells, and then culture the transformant cells in a medium containing an appropriate concentration (0.1-5%) of methanol to obtain a mutant strain using methanol as the sole carbon source and secrete and produce HSA in the medium. Someone has carried out a series of comparative experiments on the physicochemical properties, spatial structure and pharmacological properties of the recombinant HSA produced by the expression of Pichia pastoris and natural HSA, and the results show that the two are almost identical (see KazuoI Kegayc, Anal, Chem, 69:1986-1991 , (1997)). In addition, European Patent Applications Nos. 0339455, 0123544, 0248637 and 0251744 also disclosed methods for expressing human serum albumin using yeast hosts and corresponding purification methods for the products. Therefore, these research results make it possible to produce high-purity HSA with high yield by using recombinant DNA technology.

In order to further improve the expression yield of HSA in yeast cells, some laboratories have also conducted a series of in-depth studies on the signal peptide, promoter sequence of HSA, and the Cowpe number of the HSA gene. For example, European Patent Applications 0329127 and 0319641 disclose synthetic HSA signal peptide nucleotide coding sequences, respectively. These signal peptide nucleotide coding sequences are linked between the promoter (such as AOX1, AOX2, or GAL1, 10) sequence and the DNA sequence encoding preproHSA to improve the ability of the yeast host to express and secrete HSA. European patent application 0506040 discloses a mutant AOX2 promoter obtained by causing partial deletion or substitution in the nucleotide sequence of the natural AOX2 promoter, or adding a new nucleotide sequence therein.

On the other hand, while trying to improve the expression efficiency of yeast cells or other host systems, in-depth research has also been carried out on the purification method of recombinantly produced HSA. For example, in terms of cultivating HSA producing bacteria, it is also disclosed to add fatty acid (European patent application 0504823), glucose (Japanese patent publication JP-A219561/1989), histidine (U.S. Patent 5612197) in the culture medium to improve the production of bacteria Growth rate and method for improving HSA yield; In the presence of acetyl tryptophan (1-100mM) or other carboxylates, heat (50-70°C) process HSA-containing culture supernatant to purify HSA method (USA Patent 5132404); Combined use of ultrafiltration, heat treatment, acid treatment and ultrafiltration, and then use cation exchanger, hydrophobic chromatography carrier and anion exchanger to process, and obtain the method of pure HSA through salting out (US patent 5440018) . In addition, it also discloses a method for cultivating recombinant host cells expressing HSA in the presence of amine compounds to suppress the color generation of recombinant HSA (rHSA) products (US Patent 5369020); and using reducing agents (such as sulfhydryl compounds), Or use a cation exchanger to remove free polysaccharides, and then heat-treat rHSA to decolorize the method (European patent application 0658569).

As mentioned above, yeast cell expression system, especially methylotrophic yeast expression system is the preferred expression system for large-scale fermentative production of HSA. Although many improvements have been made in the prior art for the construction of recombinant vectors, the modification of regulatory elements for gene expression, and the culture conditions of recombinant cells, there are no necessary modifications to the HSA coding sequence that have been seen so far. , by changing the structure of HSA gene to further improve its expression efficiency and level in methylotrophic yeast.

Therefore, an object of the present invention is to provide a synthetic human serum albumin-encoding nucleotide sequence substantially consisting of yeast-biased codons and suitably modified, and functional equivalents thereof.

According to a preferred embodiment of this object of the present invention, wherein said modification includes the intron splice site of the HSA coding sequence, the transcription termination sequence of the non-transcription termination region, the long repeat sequence, the internal promoter sequence and Modification of the ribosome binding site.

According to a preferred scheme of this purpose of the present invention, wherein said HSA coding sequence is through respectively synthesizing three groups of 8 nucleotide chains respectively, and then splicing into three nucleotide fragments with the synthetic technology based on polymerase chain reaction, After cloning and correcting individual base errors, they are obtained by serial connection with each other.

According to a preferred version of this object of the present invention, wherein said HSA coding sequence has the nucleotide sequence shown in Figure 1A (SEQ ID NO1).

Another object of the present invention is to provide a recombinant expression vector comprising the above-mentioned HSA coding sequence.

According to a preferred scheme of this purpose of the present invention, wherein said recombinant expression vector comprises one or more by (1) the promoter region of the methanol response gene of methylotrophic yeast, (2) coding cerevisiae cerevisiae α mating factor (AMF) prepro sequence and nucleotide sequence encoding HSA, (3) expression of a transcription terminator functionally active in methylotrophic yeast, and (4) at least one selectable marker gene and bacterial origin of replication box.

Another object of the present invention is to provide methylotrophic yeast cells transformed with the above-mentioned recombinant expression vector carrying the HSA coding sequence.

According to a preferred embodiment of this object of the invention, said methylotrophic yeast is a Pichia pastoris cell.

According to a preferred embodiment of this object of the present invention, said yeast cells may have a Mut ⁺ or Mut ^- phenotype.

Another object of the present invention is to provide a method for producing human serum albumin by recombinant DNA technology, the method comprising culturing the nucleotides comprising encoding human serum albumin prepared by the above method under conditions suitable for expressing the desired protein Sequence the yeast host cells, then recover and purify the desired human serum albumin from the culture.

Another object of the present invention is to provide the human serum albumin produced by the above method, substantially having the amino acid sequence shown in SEQ ID No:2.

Figure 1A shows the synthetic nucleotide sequence encoding full-length human serum albumin and its deduction

The amino acid sequence of human serum albumin of ;

Figure 1B shows a single sequence in the full-length human serum albumin nucleotide coding sequence shown in Figure 1A

  Endonuclease cutting site:

Figure 2 shows the synthesis strategy of the nucleotide sequence encoding human serum albumin;

Figure 3 shows the nucleotide sequence (upper line) and the deduced amino acid sequence (lower line) of the HSA-I fragment

OK);

Figure 4 shows the construction of the recombinant plasmid pBKS/HSA-I;

Figure 5 shows the nucleotide sequence (upper column) and the deduced amino acid sequence of the HSA-II fragment

(downstream);

Figure 6 shows the construction of the recombinant plasmid pBKS/HSA-II;

Figure 7 shows the nucleotide sequence (upper line) of the HSA-Ⅲ fragment and the amino acid sequence deduced therefrom

column(lower row);

Figure 8 shows the construction of the recombinant plasmid pBKS/HSA-Ⅲ;

Figure 9 shows the construction of the recombinant expression vector pBKS/HSA;

Figure 10 shows the construction of the recombinant expression vector pPIC9/HSA;

Figure 11 shows the sequence of the junction region where the HSA nucleotide coding sequence is connected to the expression vector pPIC9;

Figure 12 shows HSA-Ⅰ, HSA-Ⅱ, HSA-Ⅲ fragments and full-length HSA DNA fragments

1% agarose gel electrophoresis pattern;

Figure 13 shows the time when the synthetic HSA gene is expressed in Pichia pastoris strain GS115

The interim process, in which the lanes 1-8 respectively show the transformation of Pichia pastoris cells

 After adding methanol to the medium to induce HSA expression, 0, 24, 24, 48, 72, 96,

120 and 148 hours culture supernatants were collected and run on 10% polyacrylamide gel

Results of the analysis;

Figure 14 shows the result of immunoblotting analysis done to HSA expression product, wherein each swimming lane

The sample materials are A: natural HSA; B: GS115 without recombinant plasmid

Culture supernatant of yeast cells; C: GS115 yeast transformed with pPIC9/HSA

The culture supernatant of the cells.

One of the most prominent advantages of yeast is that it can secrete heterologous proteins into the medium for the production of heterologous proteins by recombinant DNA technology, especially for human serum albumin, which requires extremely high purity and is rich in sulfhydryl groups in the molecule. product. Therefore, the use of yeast expression system can obtain the secreted product with higher initial purity, thereby simplifying the purification steps. Especially for sulfhydryl-rich HSA, another convincing reason for establishing a yeast expression system capable of secreting expression products into the medium is that HSA products are more likely to maintain their accurate four-dimensional structure through disulfide bond formation. Secretory pathways of cells and extracellular media are capable of supporting an oxidative environment for disulfide bond formation (Smith, et al., Science 229:1219 (1985)). In contrast, the cytosol is a reducing environment that cannot form disulfide bonds. After cell disruption, if disulfide bonds are formed too quickly, random disulfide bonds can be generated. Therefore, it is best to produce disulfide-rich proteins such as HSA via the secretory pathway.

In order to overcome some of the problems associated with S. cerevisiae, such as the loss of plasmid selectable markers and issues of plasmid distribution, copy number, and stability under high-density fermentation conditions, methylotrophic yeasts such as Pasteurbi Yeast expression system for red yeast. A key feature of this system is that it utilizes the methanol-regulated alcohol oxidase-1 (AOX1 ) gene derived from Pichia pastoris as a promoter to drive the expression of exogenous genes (see European Patent Application 183071). Another feature of this expression system is that the expression cassette can be stably integrated into the cell chromosome, thereby significantly reducing the chance of vector loss.

Prior to the present invention, not only Pichia pastoris had been used to successfully produce, for example, hepatitis B surface antigen (Cregg et al., Bio/Technology 5:479 (1987)), lysozyme and invertase (Digan et al. Developmetsin Industrial Microbiology29:59(1988); Tschopp et al., Bio/Technology5:1305(1987)), and epidermal growth factor (International Patent WO90/10697), and have also been used in methylotrophic yeast, especially Pasteurbi Human serum albumin was successfully expressed in Rhodotorula (European Patent Application 0344459). The HSA gene sequence used in the prior art is basically based on the previously isolated and sequenced HSA gene sequence by Lawn et al. and Dugaiczyk et al. However, the inventor's early research found that although the HSA gene is more suitable for expression in eukaryotic organisms including yeast, considering human cells and lower eukaryotic organisms such as yeast cells in structure and protein biosynthesis mechanism For example, there are great differences in codon usage frequency, initiation and transcription efficiency of gene transcription, RNA splicing and translation efficiency, and secretion of translation products, so it is necessary to modify the natural HSA gene to make it more suitable for It is stably present in yeast systems, especially Pichia cells, and is more efficiently transcribed and translated.

In addition, some studies have shown that different organisms have large differences in codon usage frequency, which directly affects translation efficiency and expression level. If the heterologous gene is properly modified to make the codons of the gene to be expressed more suitable for the codon usage frequency of the selected expression system, it will definitely help to improve the overall expression efficiency of the foreign gene. Kerlak et al. (Bio/Technology 8:939-42, (1990)) modified the Bacillus thuringiensis (B.t) beta endotoxin gene according to the codon usage frequency of dicotyledonous plants and transformed the gene into dicotyledonous plants such as cotton cells As a result, the expression level of the modified B.t endotoxin gene in cotton is 50-100 times higher than that of the natural B.t endotoxin gene. On the contrary, Hoekeman et al. cloned the phosphoglycerol kinase gene into a yeast expression vector, and replaced some of the yeast-biased codons at its 5' end with rare codons, which resulted in a reduction of its expression efficiency in yeast cells by about 10 times .

The inventor fully considered the impact of codon usage frequency on gene expression and the possible unfavorable factors of the natural HSA coding sequence, and comprehensively modified and re-synthesized the HSA gene. To this end, firstly, the full-length human serum albumin nucleotide coding sequence was redesigned and synthesized according to the codon usage frequency of Saccharomyces cerevisiae whose codon usage frequency is substantially the same as that of Pichia pastoris. Compared with the natural HSA gene sequence, 155 bases in the synthesized gene sequence of the present invention are replaced by other bases, and thus 141 codons are changed. The codon usage frequency of the HSA gene synthesized by the present invention is shown in Table 1 below.

Table 1: Codon Usage Frequency of Synthetic HSA Genes a amino acid usage frequency a amino acid usage frequency a amino acid usage frequency a amino acid usage frequency TTTTTCTTTATTGCTTCTCCTACTGATTATCATAATGGTTGTCGTAGTG phepheleuleuleuleuleuleuileileilemetvalvalvalval 1913121810110114416159611 TCTTCCTCATCGCCTCCCCCACCGACTACCACAACGGCTGCCGCAGCG serserserserproproproprothrthrthralaalaalaalaala 854183112117732715164 TATTACTAATAGCATCACCAAACAGAATAACAAAAAGGATGACGAAGAG tyrtyrOCHAMBhishisglnglnasnasnlyslysaspaspgluglu 991111514698322826114221 TGTTGCTGATGGCGTCGCCGACGGAGTAGCAGAAGGGGTGGCGGAGGG cyscysOP Atrpargargargargserserargargglyglyglygly 2213-1312152145923-

In order to facilitate the connection of the synthetic HSA gene of the present invention to various cloning vectors or expression vectors, we added two single restriction sites at the 5' and 3' ends respectively, that is, BamHI and XhoI enzymes were added at the 5' end EcoRI and ClaI restriction sites were added to the 3' end, among which BamHI and ClaI cutting sites were used to connect the cloning vector, while XhoI and EcoRI were used to connect the expression vector. In addition, in order to ensure the effective utilization of restriction sites, we added three protective bases (GGC and AGG, respectively) at the 5' end and 3' end of the synthetic gene sequence. Furthermore, in order to facilitate the connection of the synthetic HSA gene to the C-terminus of the signal peptide, an amino acid sequence was added to the N-terminus of the gene as a signal peptide recognition and cleavage site. As an essential regulatory element of the HSA gene, its C-terminus has a repeated stop codon sequence to ensure the correct termination of the mRNA translation process. The above modifications lead to the total length of the synthesized HSA gene sequence of the present invention being about 1797bp.

On the other hand, in order to further improve the expression efficiency of the HSA gene synthesized by the present invention in methylotrophic yeast, especially Pichia pastoris, the following several possible gene transcriptions of the nucleotide sequence of the gene Necessary modifications, alterations, substitutions or deletions have been made to sequences or other parts that adversely affect translation efficiency and/or translation efficiency.

1. Intron splicing sequence: Eukaryotes have the ability to splice and process gene transcription products to form mRNA without introns. It is now known that the splicing of introns is mediated by special sequences. If there are corresponding splicing sites in the DNA sequence, such sequences can be cut out from the sequence as introns during gene transcription. resulting in incomplete transcripts. Therefore, in the design and synthesis of genes, it is necessary to check and exclude such base sequences.

2. Transcription termination sequence: If there is an additional transcription termination sequence at the non-transcription termination site within the full-length gene sequence, it may lead to premature or wrong termination of transcription, resulting in incomplete mRNA. In the yeast expression system, sequences known to be associated with transcription termination include: (1) ATTATTTTAT; (2) TTTTTATA; (3) TAG...(T-rich)...TA(T)GT...(AT-rich)...TTT . Therefore, when designing and synthesizing the HSA gene, these sequences, especially AT-rich sequences, must be excluded.

3. Long repeat sequences: Repeat sequences, especially long repeat sequences (including forward and reverse repeat sequences) may form a strong secondary structure, which is not conducive to the smooth progress of gene transcription and translation, and may give Splicing of the full-length gene poses difficulties. Therefore, when a repeated sequence of more than seven bases appears in the sequence, it needs to be excluded by changing the involved codons without changing the encoded amino acid.

4. Internal promoter sequences and ribosome binding sites: The gene sequence may contain undesired promoter sequences that drive the internal transcription process of the gene. These sequences may interfere with the normal transcription of the full-length gene, so they must be removed. In addition, if there are redundant ribosome binding sites inside the gene, it is also possible to initiate the translation process inside the gene, resulting in incomplete protein expression products. Therefore, the TATA box promoter sequence and the GGAGG ribosome binding site were excluded in the synthetic HSA gene sequence of the present invention.

In addition, in order to facilitate the splicing of pre-synthesized polynucleotide fragments, and facilitate the splicing of the tandem spliced full-length gene sequence with other gene transcription and/or translation regulatory elements at appropriate restriction sites or convenient insertion into the selected expression In the vector, it is also necessary to add several restriction sites or linker sequences at appropriate nucleotide positions in the gene sequence. Therefore, in addition to the BamHI and XhoI restriction sites at the 5' end and the EcoRI and ClaI restriction sites at the 3' end mentioned above, HindIII and PstI sites were added at nucleotide positions 509 and 1109, respectively. (Fig. 1B).

Therefore, the first object of the present invention is to provide a nucleotide sequence encoding human serum albumin synthesized according to the above principles. In order to obtain the full-length nucleotide sequence encoding human serum albumin as shown in Figure 1A, three nucleotide sequences of 612bp, 562bp and 705bp were synthesized and named HSA-I, HSA-II and HSA respectively Fragment of -III. Each fragment is connected by 8 shorter oligonucleotides, and both ends of each fragment have BamHI and ClaI restriction sites for direct ligation and cloning in appropriate cloning vectors.

Complementary strands of relatively short oligonucleotide sequences can be chemically synthesized according to conventional methods known to those skilled in the art, and denatured and then annealed to obtain corresponding double-stranded DNA molecules. However, from the viewpoint of economy and convenience, it is better to use the PCR-based DNA sequence synthesis method improved by the present inventors to synthesize the HSA gene sequence of the present invention (see Example 1). According to this method, only an overlapping complementary region of ≤20 base pairs is reserved between the two complementary strands, which is enough to synthesize a double-stranded oligonucleotide sequence up to 120 bases based on a template strand. In this way, correctly connected DNA fragments up to about 900 bp can be obtained after one splicing.

Because when utilizing Oligo4.0 computer software to design each oligonucleotide chain, fully analyzed the overlapping region sequence on the adjacent two oligonucleotide chains, thereby effectively avoided the hairpin structure and in said overlapping region The formation of a strong secondary structure increases the molecular internal stability of the sequence specificity of the overlapping region. Generally, this can be achieved by altering individual bases within the overlapping region or by causing a shift in the overall overlapping region.

Figure 2 schematically shows the synthesis strategy of the HSA nucleotide coding sequence of the present invention. Briefly, three DNA fragments named HSA-I, HSA-II, and HSA-III were first synthesized with lengths of 612bp, 362bp, and 705bp (see Figures 3, 5, and 7). Each of these fragments consisted of 8 oligonucleotide sequences. Then these three fragments were respectively cloned into the pBKS plasmid vector carrying the M13 phage DNA replication origin, ampicillin resistance gene and multiple cloning site, and respectively containing T3 and T7 phage promoters on both sides of the multiple cloning site, to obtain Three recombinant plasmids were named pBKS/HSA-I, pBKS/HSA-II and pBKS/HSA-III respectively (see Figures 4, 6, and 8). After DNA was extracted from these plasmids and the nucleotide sequences were determined respectively, errors in individual bases were corrected in an appropriate way according to the above principles. After the base sequence of each fragment is determined, use the preset HindⅢ and PstI restriction sites at both ends of each fragment to connect them to the linearized plasmid pPKS after cutting with BamHI and ClaI. Under the action of _T4 DNA enzyme The recombinant plasmid pBKS/HSA was obtained after re-circularization (Fig. 9). After being transformed into Escherichia coli DH5α for several passages, DNA was extracted from the proliferating bacterial cells, and the nucleotide sequence of the full-length HSA gene was determined by the dideoxy chain termination method. The results showed that the synthesized HSA of the present invention can be stably retained in the subsequent coli cells without any sequence mutations and/or deletions.

The expression vector used to express the HSA gene synthesized by the present invention can be any vector that carries expression control elements suitable for expressing foreign genes in yeast, and can be inserted with a high copy number in yeast, especially methylotrophic yeast, For example pPIC9 (Invitrogen), pPIC3 (Invitrogen), pHIL-D2 (Invitrogen), yYJ32 (NRRL B-15891), pJDB207 (Amershan), pJDB219 (Amershan), PG4.0 (NRRL B-15868), for the present invention purpose, among which pPIC9 is preferred.

For example, EcoRI and XhoI double enzyme digestion can be used to linearize the pPIC9 plasmid and ligate it with the HSA gene fragment obtained from pBKS/HSA after the same double digestion, and then use the resulting ligation mixture to transform appropriate Escherichia coli cells. After culturing on LB medium plate at 37°C and screening the transformed recombinant cells, the plasmid DNA was extracted and the recombinant plasmid was identified according to the fragment size.

Therefore, the present invention further relates to a recombinant expression vector containing at least one expression cassette, wherein said expression cassette contains the following DNA sequences sequentially in the direction of the reading frame of transcription:

(i) the promoter region of the methanol-responsive gene of methylotrophic yeast;

(ii) DNA sequences encoding the preprosequence of Saccharomyces cerevisiae alpha mating factor (AMF) and the sequence encoding the HSA polypeptide, and

(iii) A transcription terminator functionally active in methylotrophic yeast.

This DNA fragment can be transformed into a methylotrophic yeast such as Pichia pastoris as a linear fragment flanked by a DNA sequence having sufficient homology to the target gene to achieve the expression of the DNA fragment on the yeast chromosome. integration in . In this case, integration is achieved by homologous recombination of homologous sequences in the recombinant expression vector with the yeast target genome at appropriate sites in the yeast target genome. Alternatively, the DNA fragment can be part of a circular plasmid that can be cut linearly to facilitate integration, and the fragment can be added at a site of homology between the host and plasmid sequences to achieve its integration in the host genome. integrate. Linearization of the vector will facilitate the integration of the exogenous gene and often inserts into the host chromosome at the linearization site.

An expression cassette for transformation of methylotrophic yeast cells containing, in addition to a methylotrophic yeast methanol-responsive promoter and an HSA-encoding DNA sequence (HSA gene), a cleavage recognition site for the Saccharomyces cerevisiae preprosequence in coding reading frame (ie Glu-Lys-Arg coding sequence), and a transcription terminator sequence that can function in methylotrophic yeast.

A preferred promoter region for driving expression of the HSA gene is derived from the methanol-inducible or regulated alcohol oxidase (AOX) gene of Pichia pastoris. Pichia pastoris is known to contain alcohol oxidase 1 (AOX1) and alcohol oxidase II (AOXII) genes. The coding parts of the two AOX genes share great homology at the level of DNA and deduced amino acid sequences, and share common restriction enzyme sites. The proteins expressed by the two genes have similar enzymatic properties, but the promoter of the AOX1 gene is more effective. Therefore, for the purpose of expressing HSA, it is preferred to use AOX1 (Ellis et al., Mol. Cell. Biol. 5:1111 (1985)).

Yeast alpha mating factor or alpha factor is a 13 amino acid peptide secreted by cells of the "alpha" mating type. It acts on cells of relative "a" mating type to promote efficient conjugation between the two types of cells, resulting in "alpha-a" diploid cells (Thorner et al., The Molecular Biology of The Yeast Saccharomyces, Cold Spring Harbor Laboratory, Cold Spring Harbor, 143 (1981)). The AMF preprosequence is the leader sequence contained in the AMF precursor and includes the Glu-Lys-Arg coding sequence required for proteolytic processing and secretion. The AMF prepro sequence (including the Leu-Lys-Arg coding sequence) is a 258bp fragment.

The expression cassette for expressing HSA in methylotrophic yeast according to the invention also contains a transcription terminator, which has a subfragment encoding a polyadenylation signal and a polyadenylation site, and/or is derived from a promoter A subfragment that initiates transcription and provides a transcription termination signal.

The above-mentioned functional regulatory sequences (non-structural gene regulatory elements) for HSA expression and secretion of the present invention have generally been included in the expression vector pPIC9 (Invitrogen) selected for the purpose of expressing HSA in the present invention, and wherein the transcription terminator and The origin of the promoters can be the same or different.

The expression vector according to the present invention may also include a selectable marker gene that can function in methylotrophic yeast such as Pichia pastoris. For this purpose, the selectable marker gene can be any gene that confers a phenotype on methylotrophic yeasts such as S. pastorii, thereby allowing them to be identified and conferring on them a growth ability not possessed by untransformed cells . For example, suitable selectable marker genes include a selectable marker system consisting of an auxotrophic mutant Pichia pastoris host strain and wild-type biosynthetic genes that complement the host deficiency. For example, when transforming a HIS4 ^- Pichia pastoris strain, the Saccharomyces cerevisiae or Pichia pastoris HIS4 gene can be utilized.

If the yeast host is transformed with a linear DNA fragment containing the HSA gene under the regulation of the Pichia pastoris gene promoter and the AMF sequences required for processing and secretion, the above expression cassette will be replaced by known one or two steps Technology (see Rothstein, Methods Enzymol.101: 202 (1983); Sherer and Davis, Proc. Natl. Acad. Sci., USA, 76: 4951 (1979)) is integrated into the host genome, which can be integrated with the target gene Flanking DNA sequences with sufficient homology direct the linear DNA fragments to specific loci for integration of the DNA fragments therein.

If the DNA fragment containing the synthetic HSA coding sequence of the present invention is contained in an expression vector such as a circular plasmid, one or more copies of the plasmid can be integrated at the same or different loci of the genome. Before or after the DNA fragment containing the synthetic HSA coding sequence of the present invention is integrated, the various parts of the expression cassette are operably linked to each other. With respect to the promoter, the DNA sequence encoding the AMF processing site in Saccharomyces cerevisiae, and the transcription terminator, the DNA sequence encoding HSA is located in its functional position and orientation. Thus, the HSA coding sequence can be transcribed under the control of the promoter region into a transcript capable of producing the desired HSA upon translation. Because of the presence of the AMF prepro sequence within the expression vector, the HSA product as a whole is secreted and can be isolated from the culture medium. Since the DNA fragment containing the HSA gene sequence synthesized in the present invention includes a sequence allowing it to replicate in bacteria, especially Escherichia coli, the DNA fragment can be produced in large quantities in bacteria.

According to a preferred embodiment of this purpose of the present invention, the DNA sequence encoding HSA synthesized according to the above-mentioned method of the present invention can be operably linked to a commercially available DNA sequence that is suitable for expression in methylotrophic yeast. and expression vectors of functional regulatory sequences (non-structural gene regulatory elements) that secrete foreign proteins, such as pPIC9 that can be purchased from Invitrogen. The plasmid contains 5'AOX1 promoter fragment, α-factor secretion signal, multiple cloning site region, 3'AOX1 transcription termination fragment, HIS4 ORF, 3'AOX1 fragment, COLE1 replication origin and ampicillin resistance gene. Methods for transforming methylotrophic yeast such as Pichia pastoris, and methods for cultivating methylotrophic yeast such as Pichia pastoris containing a gene encoding a foreign protein in its genome are in the art known. According to the present invention, available Cregg et al. (Mol.Cell.Biol.55:3376 (1985)), described protoplast technique or whole cell chlorination yeast transformation system (ITO, et al, Agric.Biol.Chem. 48:341(1984)) and electroporation to transform the expression vector into methylotrophic yeast cells. In cases where a single transcription unit is used, electroporation transformation is preferred.

DNA integration sites were analyzed by Southern blot analysis (Sambrook et al., Molecular Cloning, 2nded., Chapter 9, Cold Spring Harborlaboratory Press, Cold Spring, NY, USA, 1989), and analyzed by Northern blot (Sambrook et al., cited above). , Chapter 7) analysis of methanol-responsive HSA gene expression to further identify positive transformant cells.

The present invention further provides a method for producing recombinant HSA using a methylotrophic yeast expression system, the method comprising cultivating the yeast host cell comprising the nucleotide sequence encoding HSA prepared by the above-mentioned method under conditions suitable for expressing the desired protein , the HSA is then recovered and purified from the culture. For this purpose, transformed strains with the expected phenotype and genotype are grown in fermenters. For large-scale production of products based on recombinant DNA technology from methylotrophic yeasts such as Pichia pastoris, a three-stage high-density batch fermentation system is generally available. The first phase, the growth phase, involves culturing the host cells in a medium with excess glycerol as a carbon source. When grown on this carbon source, the expression of heterologous genes in the yeast host is restricted, allowing cells to proliferate without producing heterologous proteins. This is followed by a shorter glycerol-limited growth phase. After the glycerol-limited growth phase, methanol is added to induce the expression of the desired heterologous protein, which is the production phase (third phase). Figure 13 shows the time course of expression of the synthetic HSA gene of the present invention in Pichia pastoris.

According to a preferred embodiment of the present invention, the heterologous protein expression system for the production of HSA utilizes the promoter of the methanol-regulated AOX1 gene derived from Pichia pastoris. The promoter can effectively initiate gene expression and strictly accept the regulation of exogenous nutritional factors. The gene may also be the source of a transcription terminator. The preferred expression cassette of the present invention includes the Pichia pastoris AOX1 promoter operably linked to each other, the DNA encoding the AMF preprosequence (or alpha factor) of Saccharomyces cerevisiae, and the DNA encoding mature HSA fully synthesized according to the aforementioned principles herein sequence, and the transcription terminator derived from the Pichia pastoris AOX1 gene. Preferably two or more such expression cassettes are contained on a single DNA segment joined head-to-tail to generate multiple expression cassettes on a single contiguous DNA segment.

As mentioned above, in order to improve the expression efficiency and expression level of the HSA gene sequence in Pichia pastoris, the inventors fully considered certain structural features of the naturally occurring HSA coding sequence and their possible impact on the expression efficiency. adverse effects, the HSA gene was fully modified and de novo synthesized. These modifications include replacing all non-yeast biased codons in the native HSA gene with yeast biased codons, adding appropriate endonuclease sites to facilitate fragment ligation and recombination, and adding necessary protective bases and signals to sticky ends. Peptide recognition sequence and cleavage site, and additional stop codon. On the other hand, the present invention also aims at the intron splicing sequence in the HSA gene sequence, the long repeat sequence in the transcription termination sequence, the internal promoter sequence and the ribosome binding site, etc., which may affect gene transcription and/or translation efficiency. Necessary base modifications, alterations, and substitutions were carried out for the unfavorably affected portions.

The HSA gene sequence synthesized according to the above principle of the present invention can be inserted into an appropriate expression vector such as pPIC9, and the resulting recombinant vector can be used to transform appropriate Escherichia coli cells. Plasmid DNA was isolated from transformed E. coli and sequenced. Analysis results show that the synthetic HSA of the present invention is correctly connected between the 5' promoter sequence and the 3' transcription terminator sequence of the AOX1 gene carried by the expression vector, and is directly connected to the AMF prepro sequence (α Factor coding sequence) on the 3' coding sequence of the proteolytic processing site Glu-Lys-Arg (see Figure 11).

The modification and alteration of the natural HSA coding sequence by the above-mentioned synthetic gene and its correct connection in an appropriate expression vector fully ensure the effective initiation of gene expression, smooth transcription and transcription termination, and also ensure the smooth progress of the translation process of the transcript .

Preferred host cells that can be transformed with the above-mentioned expression cassettes are Pichia pastoris cells with at least one mutation complemented by a marker gene present on the transforming DNA fragment, preferably using HIS4-(GS115) or ARG4-( GS190) auxotrophic mutant Pichia pastoris strain. A DNA fragment containing one or more expression cassettes is inserted into a plasmid containing a marker gene that complements a defect in the host. The preferred expression vector of the present invention is plasmid pAO815 or pPIC9, especially pPIC9.

In order to establish Mut ^- expressing strains of Pichia pastoris, a one-step gene replacement technique can be used to integrate the transformation DNA containing the expression cassette into the host genome, and digest the expression vector with an appropriate endonuclease to generate homologs at both ends. Linear DNA fragment of the AOX1 locus (via flanking homologous sequences). The Mut ³ strain was obtained through such gene replacement. The Mut mentioned here refers to the methanol utilization phenotype. In the Mut ³ strain, the AOX gene was replaced by an expression cassette, thereby reducing the ability of the strain to utilize methanol. In this case, the yeast strain maintained its slow growth on methanol through the expression of the AOX2 gene product. Transformants in which the expression cassette has been integrated into the AOXI locus by site-specific recombination can be identified by the presence of a compensatory gene.

In order to establish a Mut ⁺ strain expressing HSA, it is preferable to transform a host cell with a circular plasmid including an expression cassette, so that the DNA fragment including one or more expression cassettes is integrated into the host genome. In this case, integration is achieved by addition at loci that share homology to one or more sequences on the transformation vector. To identify positive transformants, the site of DNA integration can be analyzed by Southern blotting, the expression of methanol-responsive HSA gene can be analyzed by Northern blotting, and secreted expression of HSA protein can be confirmed by detecting HSA product in the culture medium. Pichia pastoris strains incorporating one or more expression cassettes at the expected sites can be identified by Southern blotting. Yeast strains with increased HSA secretion yields can be identified by Northern blotting and protein product analysis (see Figure 14).

Methylotrophic yeast transformants that demonstrate expected genotypes and phenotypes are grown in fermentors, eg, in modified YNB or YPD broth, at a temperature of about 30°C. HSA can be produced fermentatively using a typical three-stage production process, starting with cells grown on an inhibitory carbon source, preferably on an excess of glycerol. This stage is to generate a population of cells in the absence of expression. Cells were then grown under conditions of limited glycerol supply. After glycerol is consumed, methanol alone or limited amounts of glycerol+methanol are added to the incubator to result in expression of the HSA gene driven by a methanol-responsive promoter. In order to ferment at a high cell density and increase the expression level of the product, a small amount of high-concentration sucrose and glucose can be added at this stage in a fed-batch culture method to avoid inhibition of cell growth by high-concentration substrates. Fatty acids, such as fatty acids with 6-18 carbon atoms, are added to the medium to improve the yield of HSA product.

The method for isolating and harvesting HSA may include heat-treating the above-mentioned culture supernatant to inactivate proteases, and using at least one material selected from the group consisting of cation exchangers, hydrophobic carriers, and activated carbon to separate HSA from coloring components to inhibit the production of the product. Color generated. Appropriate amines such as methylamine, ethylamine or propylamine can be added to the medium during the culture to suppress the color of HSA.

High-purity HSA can be purified by at least five combined methods selected from heat treatment, ultrafiltration, affinity chromatography, cation exchange chromatography, hydrophobic chromatography, anion exchange chromatography, salting out, and chelating resin treatment. In order to appropriately simplify the purification steps and reduce the production cost as much as possible under the premise of ensuring the purity of the HSA product, it is better to use sequential purification of heat treatment, ultrafiltration, hydrophobic chromatography, and anion exchange chromatography or high pressure liquid chromatography method. The yield of HSA produced by the method of the present invention is generally about 3.5 g/liter of culture solution, and the purity of the product can basically reach 99.99%.

The following examples are intended to further exemplify and describe the present invention, but these examples are not intended to limit the scope of the pending claims of the present invention in any way. Example 1: Synthesis and cloning of the full-length sequence of the HSA gene

This example describes the synthesis of the full length nucleotide sequence encoding HSA. In view of the relatively long nucleotide sequence of the full-length HSA gene, one-time synthesis may encounter operational difficulties, so the full-length gene sequence was divided into three larger nucleotide fragments and synthesized separately. These three fragments are called HSA-I (612bp), HSA-II (562bp), and HSA-III (705bp), respectively, and each of them is spliced by 8 oligonucleotide fragments. 1. Synthesis and Cloning of HSA-Ⅰ Fragment

(1) In order to synthesize the fragment HSA-I including the 5' side of the HSA gene with a length of 612bp, first use the ABI394 type DNA automatic synthesizer (Appiled Biosystem Inc.) on a solid phase carrier at 1000 Å according to the steps recommended by the instrument manufacturer合成有下示序列的8个寡核苷酸链：OA-1GGCGGATCCCTCGAGAAAAGAGATGCACACAAGAGTGAGGTTGCTCATCGGTTTAAAGATTTGGGTGAGGAAAATTTCAAAGCCTTAGTGTTAATOA-2GCGAACTCAGTGACCTCGTTAACCAACTTTACATGATCCTCAAATGGACATTGCTGAAGATATTGGGCAAAAGCAATTAACACTAAGGCTTTGOA-3CGAGGTCACTGAGTTCGCTAAAACTTGTGTTGCAGACGAATCTGCTGAAAATTGTGATAAGTCTCTGCATACCCTATTTGGTGACAAATTGTGCOA-4GCATTCGTTTCTGCCAGGTTCTTGCTTTGCACAGCAGTCGGCCATTTCACCGTAAGTTTCTCGAAGGGTAGCCGACTGTGCACAATTTGTCACCAAAOA-5CCTGGCAGAAACGAATGCTTTTTGCAGCACAAACATGACAACCCTAATCTTCCACGTTTAGTGAGACCTGAAGTTGATGTGATGOA-6TATGGGTGCCTTCTTGCAATTTCATATAAGTACTTCTTCAAAAAAGTTTCTTCATTATCATGGAAGGCTGTACACATCACATTCAACTTCAGGOA-7GCAAGAAGGCACCCATACTTCTATGCCCCGGAGCTACTGTTCTTTGCTAAACGTTATAAAGCTGCATTCACAGAATGTTGCCAAGCOA-8CCTATCGATGGCAGAAGAAGCTTTACCCTCATCTCTTAATTCGTCTAGCTTTGGCAACAGACAAGCTGCCTTATCGGCAGCTTGGCAACATTCTGTG

The respectively synthesized oligonucleotides (OA-1 to OA-8) were eluted from the solid-phase carrier column and purified by 10% polyacrylamide gel electrophoresis containing 7M urea, and recovered respectively.

According to the synthesis strategy shown in Figure 2, using the DNA synthesis technology mediated by PCR reaction, the HSA-I fragment was obtained by three-step PCR amplification from the above eight oligonucleotides. Specifically, first add OA-1 and OA-2 (10 μl each), OA-3 and OA-4 (10 μl each), OA-5 and OA-6 (10 μl each) and OA-7 and OA-8 (10 μl each). Approximately 50 l of 1×PCR buffer, 200 μM dNTPs (50 μM each), 1.5 mM MgCl ₂ and 2.5 units of Taq DNA polymerase were then added to each tube with a total reaction volume of 50 μl. Under the PCR reaction conditions of 94°C, 10 seconds; 55°C, 30 seconds; 72°C, 1 minute, after 15 PCR cycles, 4 double-stranded DNA fragments in which 8 oligonucleotide chains are connected in pairs are obtained: Fragment A, B, C, and D. Then fragments A and B, C and D were mixed in two reaction containers, and the PCR reaction was carried out again according to the above conditions to obtain further fused and amplified fragments E and F respectively. Finally, using oligonucleotides OA-1 and OA-8 as primers at both ends, the PCR reaction was performed again on the mixture of fragments E and F obtained above, thereby obtaining fragment HSA-I representing the 5' end of the HSA gene. After the fragment was separated and purified by 1.5% agarose gel, it was stored at -70°C for future use.

(2) Inoculate the Escherichia coli DH5α strain carrying the plasmid pBKS in 50ml of LB medium, and culture with shaking at 37°C for 15 hours. After culturing, the cells were collected by centrifugation (5000 rpm, 5 minutes), and the plasmid pBKS for cloning the synthetic HSA fragment was prepared by alkaline lysis. To do this, first suspend the cell pellet in 5ml solution I (50mM sucrose, 25mM Tris, 10mM EDTA, pH 8.0), then add 10ml fresh solution II (0.2NaOH, 1% SDS) to the suspension, mix well Place in an ice-water bath for 10 minutes, then add 7.5ml of solution III (3MKAc, pH 4.8), mix again and place in an ice-bath for 15 minutes, centrifuge (12000rpm, 10 minutes) to collect the precipitate. Wash the cell pellet with 70% ethanol and resuspend in 1 ml TE buffer. To the resulting solution was added RNase A (Promega) (200 µg/ml) and incubated at 37°C for 30 minutes. Then use phenol successively; Anhydrous ethanol to precipitate it. After centrifugation, the precipitate was washed with 70% ethanol and redissolved in 0.1 ml TE buffer to obtain pBKS plasmid DNA. Finally, the plasmid DNA was digested with restriction endonucleases BamHI and ClaI (2 units each), separated and recovered by 1% agarose electrophoresis to obtain a purified linearized pBKS vector.

(3) Digest the HSA-I fragment (4 μg) prepared above with BamHI and ClaI double enzymes (1 unit each), and combine the digested HSA-I fragment product (1 μg) with the pBKS plasmid digested in the same way as above (about 4 μg) were mixed, and reacted in a water bath at 16° C. for 17 hours in the presence of T4 DNA ligase (3 units). After the reaction was completed, the obtained ligation mixture was used to transform the cell suspension of competent Escherichia coli DH5α strain (OD ₆₀₀ was about 0.6) according to conventional methods. The pBKS empty plasmid was used as a negative control in the transformation reaction. The bacterial cell suspension was plated on a solid LB plate supplemented with ampicillin (100 μg/ml), incubated overnight at 37°C, and the transformed positive colonies were screened out, and plasmid DNA was prepared. The DNA was digested with BamHI and ClaI, and the recombinant plasmid pBKS/HSA-I with the HSA-I fragment linked in the correct direction was preliminarily identified according to the size of the obtained DNA fragment.

(3) Cultivate the preliminarily identified positive strain carrying the recombinant plasmid pBKS/HSA-I on a solid medium supplemented with ampicillin (100 μg/ml), and prepare DNA from it according to the above method, and use the DNA as the following sequencing reaction template. Template DNA (2 μg) was denatured according to a known method, and then sequencing primers and annealing buffer (2 μl each) were added to complete the annealing reaction, and the known Sanger dideoxy chain termination method (Sambrook et al., Molecular Cloning, 2nd Edition Chapter 13, Cold Spring Harbor laboratory Press, Cold Spring, NY, USA, 1989) detected the nucleotide sequence of the denatured double-strand DNA template, and the sequencing results showed that all five clones contained varying numbers of bases Deletions and substitutions. To this end, use the enzyme cutting sites at the appropriate positions in the HSA-I series to cut out small fragments without base errors and reconnect them with small fragments with correct sequences in other clones to obtain a base-removed fragment. HSA-I fragment. In correcting base errors in the HSA-I fragment, two internal single restriction sites, HaeIII at nucleotide position 282 and ScaI site at nucleotide position 431, were selected and used. 2. Synthesis and Cloning of HSA-Ⅱ Fragment

按照上文1(1)中所述合成HSA-Ⅰ片段的相似方法，首先合成具有下示8个寡核苷酸：OA-9GGCGGATCCGAGGGTAAAGCTTCTTCTGCCAAACAAAGATTGAAGTGCGCTAGTCTACAGAAATOA-10AAACTCGGCTTTGGGAAATCTCTGGCTCAGGCGAGCTACAGCCCATGCCTTAAAAGCTCTTTCTCCAAATTTCTGTAGACTAGCGOA-11TTCCCAAAGCCGAGTTTGCAGAAGTTTCCAAGTTAGTGACGGATTTGACCAAAGTCCATACGGAATGTTGCCATGAGATTTGCTTGAATGTGCOA-12CTTCAGTTTACTCGAGATCGAATCTTGATTTTCACAGATATACTTGGCCAAGTCCGCCCTGTCATCAGCACATTCAAGCAAATCTCCOA-13CGATCTCCAGTAAACTGAAGGAATGTTGTTAAAAACCTCTTTTGGAAAAATCCCACTGCATTGCCGAAGTGGAAAACGATGAGATGCCOA-14CCTTTGCCTCAGCGTAGTTTTTGCAAACATCCTTACTTTCAACAAAATCAGCAGCTAATGATGGCAAGTCAGCAGGCATCTCATCGTTTTCCOA-15CTACGCTGAGGCAAAGGATGTCTTCTTGGGCATGTTTTTGTATGAATACGCAAGAAGGCATCCAGATTACTCTGTCGTGCTGTTCCCCCGCTGAOA-16CCTATCGATACATTCATGAGGATCTGCAGCGGCACAGCACTTCTCTAGAGTGGTTTCATATGTCTTGGCAAGTCTCAGCAACAGACCACA

Then the 8 oligonucleotides (OA-9 to OA-16) were ligated to have the 5' end BamHI and the 3' end ClaI restriction enzyme-cut site, a double-stranded HSA-II fragment with a length of 564bp. This fragment represents the middle part of the HSA gene sequence. Similarly, the synthesized HSA-II fragment was purified and cloned according to the methods described in 1(2) and above, and the recombinant cloning vector pBKS/HSA-II containing HSA-II was prepared. The construction of the recombinant cloning vector pBKS/HSA for cloning and characterization purposes is schematically shown in Figure 6 . Then, according to the method described in 1(3) above, 5 Escherichia coli clones carrying pBKS/HSA-II were also taken, and the DNA was isolated and sequenced. Sequence analysis results showed that only one clone had a single base error in the base sequence of the five clones analyzed, but the error did not lead to changes in the amino acid sequence. 3. Synthesis and Cloning of HSA-Ⅲ Fragment

按照上文1(1)所述的合成HSA-Ⅰ片段的方法，首先合成具有下示序列的8个寡核苷酸：OA-17CGCGGATCCGTGCCGCTGCAGATCCTCATGAATGTTATGCCAAAGTTTTCGATGAATTTAAACCTCTTGTGGAAGAGCCACAAAATTTAATTAAACAAAATTGTGAGOA-18GGAGTTGACACTTGGGGTACTTTCTTGGTGTAACGAACTAACAGCGCATTCTGGAATTTGTATTCACCAAGTTGCTCAAAAAGCTCACAATTTTGTTTAATOA-19CCCCAAGTGTCAACTCCAACTTTGGTAGAGGTCTCAAGAAACCTAGGTAAAGTGGGTAGCAAGTGTTTGTAAACATCCTGAAGCAAAAAGAATGCOA-20GACTCTGTCACTTACTGGCGTTTTCTCATGCAACACACATAACTGGTTCAGGACAACGGATAGATAGTGTTCTGCACATGGCATTCTTTTTGCTTCAGGOA-21CCAGTAAGTGACAGAGTCACCAAGTGCTGCACAGAATCTTTGGTTAACAGGCGACCATGCTTTTCAGCTCTGGAAGTCGATGAAACATACGTTCCCAAAGAGTTTAATGCOA-22GTGTTTCACTAGCTCAACTAGTGCAGTTTGTTTCTTGATTTGTCTTTCCTTCTCAGAAAGTGTACATATATCTGCATGGAAGGTGAAAGTTTCAGCATTAAACTCTTTGGGOA-23GTTGAGCTAGTGAAACACAAGCCGAAGGCGACTAAAGAACAACTGAAAGCTGTTATGGATGGATGATTTTCGCGGCTTTTGTAGAAAAGTGTTGTAATGCTGACGOA-24CCTATCGATCAAGAATTCCTATTATAAACCTAAGGCAGCTTGAGATGCAGCAACTAGTTTCTTACCTTCCTCGGCAAAGCAAGTTTCCTTATCGTCAGCCTTAGAAGAC

Then, the eight oligonucleotide chains (AO-17 to OA-24) were ligated into 5'-end BamHI and 3'-end ClaI enzymes through PCR amplification three times as described above. site, a double-stranded HSA-Ⅲ fragment with a length of 705 bp. This fragment represents the 3' portion of the HSA gene sequence. Purify and clone the synthesized HSA-III fragment by the similar method described in 1(2) above, and prepare the recombinant cloning vector pBKS/HSA-III containing HSA-III. The construction of the recombinant vector pBKS/HSA-III for purification, cloning and characterization purposes is shown schematically in Figure 8 . Then, according to the method described in 1(3) above, 5 Escherichia coli clones carrying pBKS/HSA-III were also taken, and the DNA was isolated and sequenced. Sequence analysis results showed that all 5 clones had individual wrong bases. To this end, subfragments with correct bases were substituted for fragments with incorrect bases as described in 1(3) above. In correcting the base error of the HSA-III fragment, the three subfragments were rejoined into the complete HSA-III fragment by means of the StyI site at nucleotide position 216 and the Hap-I site at nucleotide position 373 . 4. Preparation and cloning of full-length HSA gene

Basically, the recombinant cloning vector carrying the full-length HSA gene sequence was prepared according to the above-mentioned method for cloning each fragment of the HSA coding sequence. First, use BamHI and HindIII, HindIII and PstI, and PstI and ClaI to cut out the fragments HSA-I, HSA-II and HSA from the corresponding recombinant plasmids pBKS/HSA-I, pBKS/HSA-II and pBKS/HSA-III, respectively. -Ⅲ. After the respective fragments were purified, they were mixed together in equal amounts, and then the plasmid pBKS linearized with BamHI and ClaI double enzymes was added to the mixture of the obtained fragments. Figure 12 shows the 1% agarose gel electrophoresis patterns of HSA-I, HSA-II, HSA-III fragments and full-length HSA DNA fragments. The connection of the four fragments was completed according to the above reaction conditions, and after recircularization, screening and sequence analysis, a recombinant plasmid named pBKS/HSA was obtained. Further full-length sequence analysis showed that the obtained HSA gene sequence had a full-length nucleotide sequence as shown in FIG. 1 .

The result of the genetic stability experiment shows that after the DH5α bacterial strain of the recombinant plasmid pBKS/HSA obtained by the above-mentioned method is cultured, the plasmid DNA carried in the obtained 10 single clones has the exact same DNA sequence as the original clone, indicating that the present invention The synthetic HSA gene sequence has good genetic stability.

Embodiment 2: Construction of recombinant expression vector

This example describes the construction of a recombinant expression vector for expression of HSA in methylotrophic yeast. Generally speaking, the construction of the expression vector for the purpose of the present invention can be accomplished according to standard methods known in the art (such as referring to Sambrook et al, Molecular Cloning, A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, 1989) . The recombinant vectors for expressing the fully synthetic HSA gene of the present invention can be constructed according to the methods described in European Patent Application 0344459 and International Patent Application WO90/10697.

For convenience, as an alternative method, we used the commercially available plasmid pPIC9 (Invitrogen) suitable for the methylotrophic yeast system to construct a recombinant expression vector for secreting and expressing HSA. Plasmid pPIC9 contains the necessary regulatory sequences (non-structural gene regulatory elements) suitable for expression and secretion of foreign proteins in methylotrophic yeast, including AOX1 promoter fragment, α factor secretion signal (AMF prepro sequence), multiple cloning site point, 3'AOX1 transcription termination fragment, HIS4-ORF, 3'AOX1 fragment, ColE1 origin of replication, and ampicillin resistance gene. The use of this vector also has the advantage that it is easier to test the secretion and expression of the synthesized HSA gene of the present invention in methylotrophic yeast.

For this purpose, at first the Escherichia coli carrying the plasmid pPIC19 was shaken and cultured at 37°C for 15 hours, and after cultivation, the cell sediment was collected by centrifugation (5000rpm, 5 minutes) and the plasmid DNA was prepared by alkaline lysis (see Example 1 for the preparation method of pPKS) . The plasmid DNA was then digested with EcoRI and XhoI (3 units each). A linear DNA fragment with a length of about 8.0Kb was separated and recovered by electrophoresis on 1% agarose gel.

Mix the pBKS/HSA plasmid DNA prepared according to the above method (see Example 1) with 2 μl of multipurpose digestion buffer (Promega) and EcoRI and XhoI (3 units each) and incubate at 37° C. for 1.5 hours, and place on 1% agar The HSA gene fragment was separated and recovered by gel electrophoresis. Mix about 2 μg (10 μl) of the obtained HSA gene fragment with the pPIC9 digestion product digested with the same enzyme, and in the presence of T4 DNA ligase (3 units), incubate in a water bath at 16°C for 17 hours to complete the ligation reaction.

The resulting ligation mixture was used to transform competent E. coli DH5α cells treated with 0.1 M CaCl ₂ . Transformed colonies with ampicillin resistance (Amp ^R ) were then selected on LB plates containing ampicillin, and plasmid DNA was extracted therefrom. The resulting plasmid DNA was digested with BamHI and ClaI and initially identified by SDS-PAGE, and the recombinant plasmid pPIC9/HSA containing the HSA gene inserted in the correct position and in the correct direction was screened by DNA sequence analysis. Example 3: Transformation of yeast host strains and screening of positive transformants

This example describes the method of transforming Pichia pastoris cells with the recombinant expression vector pPIC9/HSA prepared by the above method to obtain recombinant cells stably expressing and secreting HSA.

The recombinant plasmid pPIC9/HSA was isolated from Escherichia coli DH5α carrying the pPIC9/HSA plasmid by alkaline lysis. After extraction with phenol/chloroform and precipitation with absolute ethanol, the purified plasmid DNA of pPIC9/HSA that can be used to transform Pichia pastoris strain was obtained. 10 µg of pPIC9/HSA was digested with SalI (5 units) in a 37°C water bath to cut it into linear DNA. Directly use this digestion reaction mixture to transform Pichia pastoris GS115 (HIS ^- ) strain (NRRLY-15851). The pPIC9/HSA cut by SalI can be integrated into the histidinol dehydrogenase gene in the chromosome of Pichia pastoris without destroying the AOX1 gene. Since pPIC9/HSA carries the HIS gene, the resulting transformants can be selected based on the HIS ⁺ phenotype.

Pichia pastoris GS115 [NRRL Y-15851] was inoculated in about 10 ml of YPD medium (1% yeast extract, 2% peptone, 2% glucose), and cultured overnight at 30°C with shaking. Then take 1ml of the overnight culture, culture it in 500ml of fresh YPD medium for about 12-14 hours, and centrifuge (3000rpm, 5 minutes, 4°C) to OD ₆₀₀ =1.3-1.5 to collect the cells, and resuspend them in an ice bath pre-cooled of sterile water. After washing twice more with sterile water, the cells were resuspended in 1M sorbitol solution.

Add 10 μg of linearized pPIC9/HSA plasmid DNA to 80 μl of the above-mentioned GS115 cell suspension, mix well, transfer to a pre-cooled electrotransfer tank, and place on ice for 5 minutes. Using an electroporation instrument (Invitrogen), Pichia pastoris cells were transformed by electroporation for 10 milliseconds according to the conditions recommended by the instrument manufacturer (electric field strength 1500 V/cm,). Immediately after applying the electric pulse, add 1 ml of 1 M sorbitol solution pre-cooled in an ice bath to the electrotransfer cell, and transfer the contents to a microcentrifuge tube. Take 200ul of cell suspension and spread on MD plates (1.34% YNB, 4×10 ^-5 % biotin, 0.5% methanol) and MD (1.34% YNB, 4×10 ^-5 % biotin, 1% dextrose) in the same position on the plate. At the same time, GS115 (GS115-β-gal) carrying HSA gene GS115-Albumin (HIS+Mut ^s ) galactosidase and carrying (HIS ⁺ Mut ⁺ ) strains were inoculated on MD and MM plates respectively as controls to screen for HIS ⁺ Mut ⁺ phenotype of positive transformants carrying the pPIC9/HSA recombinant plasmid. For this reason, the plates prepared above were cultured at 30C for two days, and the strains with HIS ⁺ Mut ⁺ phenotype could grow normally on both MM medium and MD medium, and the obtained strains were similar in size to the control strains. Strains with the HIS ⁺ Mut ⁵ phenotype can grow normally on MD medium, but grow very slowly or barely grow on MM medium.

Inoculate 50 HIS ⁺ Mut ⁺ monoclonal strains screened by the above method into 3ml YPD liquid medium, shake culture at 30°C for 24 hours, and then collect the cells by centrifugation. Resuspend the cell pellet in 120 μl sterile water, add 0.4 g sterile glass beads each, shake vigorously for 3 minutes to break the cells. After centrifuging to remove the cell pellet, chromosomal DNA was extracted sequentially with phenol/chloroform (1:1, total volume 100 μl) and chloroform/isoamyl alcohol (24:1, total volume 100 μl) as described above.

The chromosomal DNA prepared as above can be detected by PCR method to further screen out the positive transformants carrying the synthesized HSA gene of the present invention. To this end, add 1 μg (5 μl) of the genomic DNA of the above-mentioned transformed cells into a PCR reaction system consisting of the following components: 10X PCR reaction buffer (5 μl), about 5 μl of genomic DNA (1 μg); 100 mM dNTPs (1 μl); 5'AOX1 primer (0.1 μg/μl about 5 μl); 3'AOX1 primer (0.1 μg/μl about 5 μl); add sterile water to a total volume of 50 μl. Then add 0.25 μl Taq DNA polymerase (5U/μl), wherein the 5'AOX1 primer and the 3'AOX1 primer were purchased from Invitrogen.

The PCR reaction conditions are: heat denaturation at 94°C for 2 minutes, and then perform 25 cycles according to the following conditions: denaturation at 94°C (40 seconds), annealing at 55°C (90 seconds), extension at 72°C (1 minute), and finally 72°C Extend for 7 minutes. After the PCR reaction is completed, the PCR reaction product is electrophoresed on 1% agarose gel electrophoresis to observe whether there is an amplified HSA gene fragment, so as to further screen out transformants containing the HSA gene. The cell strain has been in accordance with the provisions of the Budapest Treaty on the Deposit of Internationally Recognized Microorganisms for Patent Procedures. The Pichia GS115 strain carrying the recombinant plasmid pPIC9/HSA will be deposited in the General Microorganism Collection Center (CGMCC) of the China Committee for the Collection of Microorganisms. Its deposit registration number is CGMCC NO.0354. Example 4: Expression of HSA gene in GS115 yeast and Western blot analysis of protein product

This example describes the expression and secretion of the HSA gene synthesized by the present invention in Pichia pastoris GS115 strain, and the western blot analysis of the expression product in the culture supernatant. The positive strains identified by the method described in Example 3 were inoculated in BMG medium (100 mM potassium phosphate, 1.34% YNB, 4×10 ⁻⁵ % biotin, 1% glycerol), and cultured with shaking at 30° C. for 18 hours. Cells were collected by centrifugation and resuspended in BMM medium (100 mM potassium phosphate, 1.34% YNB, 4× ^10-5 % biotin, 0.5% methanol) at 30°C for shaking culture. 1. Samples were taken at 144 hours, and all samples were analyzed by SDS-PAGE. The results showed that, except for 0 hour, the samples at all other time periods showed a band with a molecular weight of about 66KD, and the band changed from light to dark with the extension of expression time during this time, indicating that the expression level gradually increased (Figure 13).

The HSA protein product prepared and purified according to the above method was detected by Western immunoblotting, and commercially available recombinant HSA (provided by Shanghai Biological Products) was used as a positive control, and the culture supernatant of GS115 cells carrying the pPIC9 plasmid was fermented for 48 hours solution as a negative control.

The experimental samples and control samples were separated by sodium dodecylsulfonate-polyacrylamide gel electrophoresis (SDS-PAGE), and then the protein on the gel was transferred to a nitrocellulose filter membrane by electrophoresis. The membrane was blocked with PBS solution containing 5% skimmed milk powder at room temperature for 2 hours and washed 3 times with PBS, then the filter membrane was incubated with goat anti-HSA antiserum (1:50 dilution) at 37°C for 1.5 hours. After the filter was washed 3 times with PBS and blocked with skimmed milk powder, the filter was mixed with horseradish peroxidase-coupled monkey anti-sheep secondary antibody (Cooper Biomedical Inc.) (1:3000 dilution) and incubated at 37 Incubate for 1.5 hours at °C. Then wash 3 times with 50mM Tris-HCl, pH6.8 solution, and finally add the substrate solution o-phenylenediamine (DNB) to develop color for 5-10 minutes, and brown bands appear on the filter membrane. It can be seen from the results shown in Figure 14 that the synthetic HSA gene of the present invention can express and secrete proteins with the same immunological characteristics as natural HSA in the Pichia pastoris host. Embodiment 5: Purification of HSA expression product

The pPIC9/HSA-transformed GS115 strain was cultured with shaking at 30°C for about 48 hours in a 50 ml culture flask containing modified BMGY medium, so that the OD ₆₀₀ value of the cell culture reached 4.0. Then the cells were collected and inoculated into a two-liter fermenter containing 700 ml of BMGY medium under aseptic conditions, and fermented with aeration at 30°C. The dissolved oxygen in the tank was kept at an air saturation above 20% by adjusting the ventilation speed (5 liters/minute) and the stirring speed (1500 rpm). During the whole fermentation process, commercially available Struktol J673 antifoaming agent was regularly added to control excessive bubble generation. After the glycerol was gradually consumed, the BMMY medium (700 ml) containing 0.5% methanol but no glycerol was pumped into the tank, and the fed-batch culture was continued at 30°C. The methanol concentration in the fermenter was always kept at 0.3%-0.7% during this cultivation period. The culture was collected 20 hours after fermentation, and the natural HSA standard was used as a control, and the optical density was detected after 10% polyacrylamide gel electrophoresis and staining, and the concentration of HSA in the fermentation culture was estimated to be 4.5g/L. After the fermentation is completed, the culture supernatant is collected by centrifugation (10,000rpm, 15 minutes), heat-treated at 70°C for 20 minutes, then rapidly cooled to 15°C, and centrifuged (15,000rpm, 20 minutes) to remove heat including trace proteases. After denaturing the impurities, the centrifuged supernatant was filtered through a millipore filter membrane (Millipore) with a pore size of 0.45 μ. After filtration, the supernatant was concentrated to a volume of about 0.5 L using an ultrafiltration membrane (Millipore) with a molecular weight cut off of 30,000. Then use acetic acid to adjust the pH of the solution to about 4.5, decolorize it with RBO2 decolorizing resin (5×25 cm, column bed volume about 500 ml) equilibrated with 50 mM sodium acetate buffer (pH 4.5) in advance, and wash out to the obtained clarity Ammonium sulfate was added to the solution to make the final concentration 10%, and then adsorbed on a phenyl Sepharose column equilibrated with 50mM phosphate + 10% (NH ₄ ) ₂ SO ₄ , PH6.8 buffer solution in advance, and doubled the column bed The column was washed with the above-mentioned pre-equilibrated buffer equilibrated in volume, and finally eluted with an elution buffer (200 mM PB+30% isopropanol buffer, pH 6.8) and the eluted fraction of the 280 nM absorption peak was collected. The eluate is further desalted through a G-25 gel chromatography column to obtain a high-purity HSA product that is basically suitable for clinical application.

Sequence Listing (1), General Information (I) Applicant: Shanghai Haiji Bioengineering Co., Ltd. (II) Invention Name: Production Method of Recombinant Human Serum Albumin (III) Sequence Number: 2 (IV) Correspondence Address:

(A) Contact: Li Shaoji

(B) Street: No. 192 Taiyuan Road

(C) City: Shanghai

(D) Country: People's Republic of China

(E) Zip code: 200031 (Ⅴ) Computer readable form:

(A) Media type: 3.5-inch floppy disk

(B) Computer: AST PentiumⅢ+4/66d

(C) Operating system: Windows95

(D) Software: Word97 (Ⅵ) Telecom information:

(A) Tel: 86-21-64735898

(B) Telex: 86-21-64674742 (2) Information of SEQ ID NO: 1 (I) Sequence characteristics:

(A) Length: 1758 base pairs

(B) type: nucleic acid

(C) Chain type: double chain

(D)拓扑结构：线性(Ⅱ)分子类型：   DNA(Ⅲ)序列描述：   SEQ ID NO:11GATGCACACA  AGAGTGAGGT  GCTCATCGG  TTTAAAGATT  TGGGTGAGGA51AAATTTCAAA  GCCTTAGTGT  TAATTGCTTT  TGCCCAATAT  CTTCAGCAAT101GTCCATTTGA  GGATCATGTA  AAGTTGGTTA  ACGAGGTCAC  TGAGTTCGCT151AAAACTTGTG  TTGCAGACGA  ATCTGCTGAA  AATTGTGATA  AGTCTCTGCA201TACCCTATTT  GGTGACAAAT  TGTGCACAGT  CGCTACCCTT  CGAGAAACTT251ACGGTGAAAT  GGCCGACTGC  TGTGCAAAGC  AAGAACCTGA  GAGAAACGAA301TGCTTTTTGC  AGCACAAAGA TGACAACCCT  AATCTTCCAC  GTTTAGTGAG351ACCTGAAGTT  GATGTAATGT  GTACAGCCTT  CCATGATAAT  GAAGAAACTT401TTTTGAAGAA  GTACTTATAT  GAAATTGCAA  GAAGGCACCC  ATACTTCTAT451GCCCCGGAGC  TACTGTTCTT  TGCTAAACGT  TATAAAGCTG  CATTCACAGA501ATGTTGCCAA  GCTGCCGATA  AGGCAGCTTG  TCTGTTGCCA  AAGCTAGACG551AATTAAGAGA  TGAGGGTAAA  GCTTCTTCTG  CCAAACAAAG  ATTGAAGTGC601GCTAGTCTAC  AGAAATTTGG  AGAAAGAGCT  TTTAAGGCAT  GGGCTGTAGC651GCGCCTGAGC  CAGAGATTTC  CCAAAGCCGA  GTTTGCAGAA  GTTTCCAAGT701TAGTGACGGA  TTTGACCAAA  GTCCATACGG  AATGTTGCCA  TGGAGATTTG751CTTGAATGTG  CTGATGACAG  GGCGGACTTG  GCCAAGTATA  TCTGTGAAAA801TCAAGATTCG  ATCTCCAGTA  AACTGAAGGA  ATGTTGTGAA  AAACCTCTTT851TGGAAAAATC  CCACTGCATT  GCCGAAGTGG  AAAACGATGA  GATGCCTGCT901GACTTGCCAT  CATTAGCTGC  TGATTTTGTT  GAAAGTAAGG ATGTTTGCAA951AAACTACGCT  GAGGCAAAGG  ATGTCTTCTT  GGGCATGTTT  TTGTATGAAT1001ACGCAAGAAG  GCATCCAGAT  TACTCTGTCG  TGCTGTTGCT  GAGACTTGCC1051AAGACATATG  AAACCACTCT  AGAGAAGTGC  TGTGCCGCTG  CAGATCCTCA1101TGAATGTTAT  GCCAAAGTTT  TCGATGAATT  TAAACCTCTT  GTGGAAGAGC1151CACAAAATTT  AATTAAACAA  AATTGTGAGC  TTTTTGAGCA  ACTTGGTGAA1201TACAAATTCC  AGAATGCGCT  GTTAGTTCGT  TACACCAAGA  AAGTACCCCA1251AGTGTCAACT  CCAACTTTGG  TAGAGGTCTC  AAGAAACCTA  GGTAAAGTGG1301GTACCAAGTG  TTGTAAACAT  CCTGAAGCAA  AAAGAATGCC  ATGTGCAGAA1351GACTATCTAT  CCGTTGTCCT  GAACCAGTTA  TGTGTGTTGC  ATGAGAAAAC1401GCCAGTAAGT  GACAGAGTCA  CCAAGTGCTG  CACAGAATCT  TTGGTTAACA1451GGCGACCATG  CTTTTCAGCT  CTGGAAGTCG  ATGAAACATA  CGTTCCCAAA1501GAGTTTAATG  CTGAAACTTT  CACCTTCCAT  GCAGATATAT  GTACACTTTC1551TGAGAAGGAA  AGACAAATCA AGAAACAAAC  TGCACTAGTT  GAGCTAGTGA1601AACACAAGCC  GAAGGCGACT  AAAGAACAAC  TGAAAGCTGT  TATGGATGAT1651TTCGCGGCTT  TTGTAGAAAA  GTGTTGTAAG  GCTGACGATA  AGGAAACTTG1701CTTTGCCGAG  GAAGGTAAGA  AACTAGTTGC  TGCATCTCAA  GCTGCCTTAG1751GTTTATAA(3)SEQ ID NO:2的信息(Ⅰ)序列特征：

(A) Length: 585 amino acids

(B) type: amino acid

(C) Chain type: single chain

(D) Topological structure: linear (Ⅱ) Molecular type: Protein (Ⅲ) Sequence description: SEQ ID NO:21Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu Glu Glu18Ash Phe Le u Phe le A la la I Val Ala  Gln  Tyr  Leu  Gln  Gln  Cys35pro  Phe  Glu  Asp  His  Val  Lys  Leu  Val  Asn  Glu  Val  Tlu  Glu  Phe  Ala  Lys52Thr  Cys  Val  Ala  Asp  Glu  Ser  Ala  Glu  Asn  Cys  Asp  Lys  Ser  Leu  His  Thr69Leu  Phe  Gly  Asp  Lys  Leu  Cys  Thr  Val  Ala  Thr  Leu Arg  Glu  Thr  Tyr  Gly86Glu  Met  Ala  Asp  Cys  Cys  Ala  Lys  Gln  Glu  Pro  Glu  Arg  Asn  Glu  Cys  Phe103Leu  Gln  His  Lys  Asp  Asp  Asn  Pro  Asn  Leu  Pro  Arg  Leu  Val  Arg  Pro  Glu120Val  Asp  Val  Met  Cys  Tlr  Ala  Phe  His  Asp  Asn  Glu  Glu  Thr Phe  Leu  Lys137Lys  Tyr  Leu  Tyr  Glu  Ile  Ala  Arg  Arg  His  Pro  Tyr  Phe  Tyr  Ala  Pro  Glu154Leu  Leu  Phe  Phe  Ala  Lys  Arg  Tyr  Lys  Ala  Ala  Phe  Thr  Glu  Cys  Cys  Gln171Ala  Ala  Asp  Lys  Ala  Ala  Cys  Leu  Leu  Pro  Lys  Leu  Asp  Glu  Leu  Arg Asp188Glu  Gly  Lys  Ala  Ser  Ser  Ala  Lys  Ghn  Arg  Leu  Lys  Cys  Ala  Ser  Leu  Gln205Lys  Phe  Gly  Glu  Arg  Ala  Phe  Lys  Ala  Trp  Ala  Val  Ala  Arg  Leu  Ser  Gln222Arg  Phe  Pro  Lys  Ala  Glu  Phe  Ala  Glu  Val  Ser  Lys  Leu  Val  Thu  Asp  Leu239Thu  Lys Val  His  Thr  Glu  Cys  Cys  His  Gly  Asp  Leu  Leu  Glu  Cys  Ala  Asp256Asp  Arg  Ala  Asp  Lcu  Ala  Lys  Tyr  Ilc  Cys  Glu  Asn  Gln  Asp  Ser  Ilc  Ser273Ser  Lys  Leu  Lys  Glu  Cys  Cys  Glu  Lys  Pro  Leu  Leu  Glu  Lys  Ser  His  Cys290Ile  Ala  Glu  Val Glu  Asn  Asp  Glu  Met  Pro  Ala  Asp  Leu  Pro  Ser  Leu  Ala307Ala  Asp  Phe  Val  Glu  Ser  Lys  Asp  Val  Cys  Lys  Asn  Tyr  Ala  Glu  Ala  Lys324Asp  Val  Phe  Leu  Gly  Met  Phe  Leu  Tyr  Glu  Tyr  Ala  Arg  Arg  His  Pro  Asp341Tyr  Ser  Val  Val  Leu  Leu Leu  Arg  Leu  Ala  Lys  Thr  Tyr  Glu  Thr  Thr  Leu358Glu  Lys  Cys  Cys  Ala  Ala  Ala  Asp  Pro  His  Glu  Cys  Tyr  Ala  Lys  Val  Phe375Asp  Glu  Phe  Lys  Pro  Leu  Val  Glu  Glu  Pro  Gln  Asn  Leu  Ile  Lys  Gln  Asn392Cys  Glu  Leu  Phe  Glu  Gln  Leu  Gly Glu  tyr  Lys  Phe  Gln  Asn  Ala  Leu  Leu409Val  Arg  Tyr  Thn  Lys  Lys  Val  Pro  Gln  Val  Ser  Thr  Pro  Thr  Leu  Val  Glu426Val  Ser  Arg  Asn  Leu  Gly  Lys  Val  Gly  Ser  Lys  Cys  Cys  Lys  His  Pro  Glu443Ala  Lys  Arg  Met  Pro  Cys  Ala  Glu  Asp  Tyr Lcu  Ser  Val  Val  Lcu  Asn  Gln460Leu  Cys  Val  Leu  His  Glu  Lys  Thr  Pro  Val  Ser  Asp  Arg  Val  Thr  Lys  Cys477Cys  Thr  Glu  Ser  Leu  Val  Asn  Arg  Arg  Pro  Cys  Phe  Ser  Ala  Leu  Glu  Val494Asp  Glu  Thr  Tyr  Val  Pro  Lys  Glu  Phe  Asn  Ala  Glu Thr  Phe  Thr  Phe  His511Ala  Asp  Ile  Cys  Thr  Leu  Ser  Glu  Lys  Glu  Arg  Gln  Ile  Lys  Lys  Gln  Thr528Ala  Leu  Val  Glu  Leu  Val  Lys  His  Lys  Pro  Lys  Ala  Thr  Lys  Ghu  Gln  Leu545Lys  Ala  Val  Met  Asp  Asp  Phc  Ala  Ala  Phe  Val  Glu  Lys  cys Cys Lys Ala562Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val Ala Ala579 u u Le 585Ser Le Gln ly Ala Gla

Claims (10)

1. Suitably modified synthetic nucleotide sequences encoding human serum albumin consisting essentially of the yeast-biased codon and functional equivalents thereof. 2. The nucleotide sequence according to claim 1, wherein said modifications include splicing sites for introns of the nucleotide sequence encoding human serum albumin, transcription termination sequences of non-transcription termination regions, long repeat sequences, Modification of internal promoter sequences and ribosome binding sites. 3. The nucleotide sequence according to claim 1, wherein said nucleotide sequence encoding human serum albumin is synthesized respectively in three groups of 8 nucleotide chains, and then spliced together by a synthesis technique based on polymerase chain reaction Three nucleotide fragments are obtained in series with each other after cloning and correcting individual base errors. 4. The nucleotide sequence according to claim 1, wherein said human serum albumin coding sequence has the nucleotide sequence shown in Figure SEQ ID NO:1. 5. A recombinant expression vector comprising the nucleotide sequence encoding human serum albumin according to any one of claims 1 to 4. 6. The recombinant expression vector according to claim 5, comprising one or more promoter regions of (1) the methanol responsive gene of methylotrophic yeast, (2) the preprosequence encoding Saccharomyces cerevisiae alpha mating factor (AMF) and encoding HSA (3) a transcription terminator functionally active in methylotrophic yeast, and (4) an expression cassette consisting of at least one selectable marker gene and a bacterial origin of replication. 7. A methylotrophic yeast cell transformed with the recombinant expression vector according to claim 5 or 6. 8. The methylotrophic yeast cell according to claim 7, which is a Pichia pastoris cell. 9. The methylotrophic yeast cell according to claim 7, wherein said cell has a Mut ⁺ or ^Mut- phenotype. 10. A method for producing human serum albumin by recombinant DNA technology, the method comprises cultivating the yeast host cell comprising the nucleotide sequence encoding human serum albumin prepared by the above method under conditions suitable for expressing the desired protein, and then from The desired human serum albumin is recovered and purified from the culture.

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